The present invention relates to a method for judging a resistance to the onset of bovine leukemia caused by bovine leukemia virus BLV.
The major histocompatibility antigens (MHC antigens) are molecules involved in self-nonself differentiation in the defense mechanism of the living body against infection. They are classified into Class I molecule composed of xcex1 chain and xcex22M, and class II molecule composed of xcex1 chain and xcex2 chain. A groove for trapping an antigen peptide is present on the xcex11 and xcex12 domains, and also on the xcex11 and xcex21 domains. They are featured to have the T cell receptor recognize only a fragmented peptide trapped in the groove, thereby achieve cell death (cellular immunity) by CD8+ cells which have recognized the class I antigens, as well as induce mainly antibody production (humoral immunity) by CD4+ cells which have recognized the class II antigens.
The MHC genes constitute a gene group most full of polymorphism, and the locations of pockets, shapes, sizes and properties of the peptide trapping grooves are different among haplotypes. It is considered that association conditions of the trapped fragment peptides may vary depending on these differences, which decide immune response and disease sensitivity of each individual. The correlation between the MHC haplotypes and a resistance to a disease (disease insusceptibility) or a possibility of the onset of a disease (disease susceptibility) has been reported, for example, as to human immune deficiency virus (HIV), human T cell leukemia virus (HTLV) and malaria.
As for the bovine MHC (BoLA) class II genes, existence of DQA, DQB, DRA, DRB, DNA, DOB, DYA, and DYB genes has been estimated. DRB3, inter alia, which is one of the three genes (DRB1 to B3) identified on the DRB genetic locus, has been known to encode a functional protein, and existence of 73 alleles has been revealed so far. However, there is almost no report about correlation between bovine infectious diseases and the bovine MHC (BoLA) haplotypes.
In particular, as to the bovine leukemia virus (BLV), which has the gene PX that regulates virus proliferation in the same manner as the human immunodeficiency virus (HIV) and is a retrovirus most related to HTLV-I, a research group in the United States has reported the relationship between the bovine MHC (BoLA) haplotypes and continuous lymphocytosis mainly focusing disease resistance; however, its relationship with a possibility of the onset of the leukemia has not been reported. The ratio of cattle infected by this virus (infection rate in Japan) is 10-20%, and 1-2% of the infected cattle develops extremely malignant endemic bovine leukemia to die after a long latent period of 10-15 years. Therefore, economic loss of stockbreeders caused by the virus is very serious. If a possibility of the onset of cattle after BLV infection can be evaluated by the analysis of bovine MHC (BoLA) haplotypes, it becomes possible to select beforehand disease resistant cattle for bleeding, and it is expected that extremely safe cattle breeding can be continued.
The inventors of the present invention previously analyzed the structure of DRB gene locus among the bovine MHC (BoLA) class II genes, and reported the structures of DRB3 gene (BoLA-DRB3) and the gene product thereof (Biochem. Biophys. Res. Commun., 209, pp.981-988, 1995). The inventors further studied the function of the gene and found that a portion is present, whose amino acid sequence is distinctly different between cattle developing the leukemia and cattle not developing the disease, in the gene product from the second exon (xcex21 domain) of BoLA-DRB3 showing particularly noticeable polymorphism. They also found that the amino acid substitutions directly correlated with disease susceptibility to BLV and disease resistance. On the basis of such findings, the inventors of the present invention succeeded in providing a method for judging a resistance to the onset of bovine leukemia caused by bovine leukemia virus BLV, wherein a bovine individual, in which an amino acid specified by the amino acid number 78 of the xcex21 domain of the bovine MHC Class II DRxcex2 chain is Val, is judged to have a resistance to the onset of the leukemia (International Publication WO98/3680).
An object of the present invention is to elucidate the relationship between the bovine leukemia virus (BLV) and the bovine MHC (BoLA) haplotypes, and to provide a method for convenient and accurate judgment of a resistance to the onset of leukemia of a bovine individual caused by the bovine leukemia virus (BLV) by means of genetic engineering techniques. More specifically, the object of the present invention is to provide a method for judgment which enables judgment with higher accuracy than that disclosed in the aforementioned International Publication WO98/3680. Another object of the present invention is to provide a primer set useful for the aforementioned method for judgment.
The inventors of the present invention made intensive studies to achieve the aforementioned objects, and found that Arg specified by the amino acid number 77 of the xcex21 domain of the bovine MHC Class II DRxcex2 chain especially has an important role for a resistance to the onset of bovine leukemia. The present invention was achieved on the basis of this finding.
The present invention thus provides a method for judging a resistance to the onset of bovine leukemia caused by bovine leukemia virus BLV, wherein a bovine individual, in which an amino acid of xcex21 domain of bovine MHC Class II DRxcex2 chain specified by amino acid number 74 is Glu (glutamic acid), said amino acid specified by amino acid number 77 is Arg (arginine), and said amino acid specified by amino acid number 78 is Val (valine), is judged to have a resistance to the onset of the leukemia.
The present invention further provides a method for judging a resistance to the onset of bovine leukemia caused by bovine leukemia virus BLV, wherein a bovine individual, in which an amino acid of xcex21 domain of bovine MHC Class II DRxcex2 chain specified by amino acid number 71 is Lys (lysine) or Arg, said amino acid specified by amino acid number 74 is Glu, said amino acid specified by amino acid number 77 is Arg, and said amino acid specified by amino acid number 78 is Val, is judged to have a high resistance to the onset of the leukemia. According to a preferred embodiment of the aforementioned inventions, there is provided the aforementioned method which is applied to cattle infected by the bovine leukemia virus BLV.
According to another aspect of the present invention, there is provided a method for judging a resistance to the onset of bovine leukemia caused by bovine leukemia virus BLV, which comprises the steps of:
(1) amplifying a genomic DNA isolated from a bovine individual by polymerase chain reaction (PCR) to prepare a PCR product containing a DNA coding for a part or full length of xcex21 domain of bovine MHC Class II DRxcex2 chain, and
(2) judging that the bovine individual, in which an amino acid of the xcex21 domain of the bovine MHC Class II DRxcex2 chain specified by amino acid number 74 is Glu, said amino acid specified by amino acid number 77 is Arg, and said amino acid specified by amino acid number 78 is Val in an amino acid sequence encoded by the DNA contained in the PCR product, has a resistance to the onset of the leukemia.
The present invention also provides a method for judging a resistance to the onset of bovine leukemia caused by the bovine leukemia virus BLV, which comprises the steps of:
(1) amplifying a genomic DNA isolated from a bovine individual by polymerase chain reaction (PCR) to prepare a PCR product containing a DNA coding for a part or full length of xcex21 domain of bovine MHC Class II DRxcex2 chain, and
(2) judging that the bovine individual, in which an amino acid of the xcex21 domain of the bovine MHC Class II DRxcex2 chain specified by amino acid number 71 is Lys or Arg, said amino acid specified by amino acid number 74 is Glu, said amino acid specified by amino acid number 77 is Arg, and said amino acid specified by amino acid number 78 is Val in an amino acid sequence encoded by the DNA contained in the PCR product, has a high resistance to the onset of the leukemia.
Furthermore, the present invention provides a method for conveniently selecting a bovine individual which has a high resistance to the onset of leukemia. This method comprises the steps of:
(1) amplifying a genome DNA isolated from a bovine individual by polymerase chain reaction (PCR) using DRB40 or DRB 100 as a forward primer and SRB3 as a reverse primer to prepare a PCR product containing a DNA coding for a part or full length of xcex21 domain of bovine MHC Class II DRxcex2 chain, and
(2) judging that the bovine individual, in which the PCR product is not digested by PstI and DraIII, has a high resistance to the onset of the leukemia.
In the bovine individual selected by the aforementioned method, an amino acid of the xcex21 domain of the bovine MHC Class II DRxcex2 chain specified by amino acid number 71 is Lys or Arg, and said amino acid specified by amino acid number 74 is Glu, said amino acid specified by amino acid number 77 is Arg, and said amino acid specified by amino acid number 78 is Val.